ABSTRACT Sarcoma tumor surgery is largely successful, however, positive margins occur commonly requiring repeat surgery or additional radiation, and recurrence of tumors is associated with significant morbidity. The surgical resection process is largely done through pre-surgical clinical imaging combined with visual and tactile cues during the operation. Molecular fluorescence guidance could significantly improve the success of surgery for sarcomas and other cancers by providing a direct, real-time report of the tumor margin thickness. We are well poised to develop this concept with an EGFR-targeted fluorescent probe (ABY- 029), developed under Good Manufacturing Practice (GMP) production, and just recently approved under an IND from the FDA for human testing. This pilot study will analyze the signal as a function of overlying tissue thickness to help establish the clinical interpretation of how to utilize the images from a surgical fluorescence system. The raw images will be compared with perfusion fluorescence imaging, and quantitative analysis completed on ratio imaging of the two as a guidance that would potentially provide even more information about both vascular perfusion and EGFR molecular expression. Once the signal interpretation methodology is established in Aim 1, the real signal testing and logistics of administration time will be established in human sarcoma xenograft studies. These will be used to determine the injection/imaging time sequence for the EGFR probe and the perfusion probes. The sensitivity and ability for multi-wavelength imaging will be established in this pilot phase trial, immediately before initiating human sarcoma studies to gauge detection ability in surgical studies. This is a very small pilot phase application that will leverage an enormous resource of collaborators and infrastructure at the Dartmouth-Hitchcock Medical Center (Dept. of Orthopaedics and Sarcoma Program), the Norris Cotton Cancer Center (supporting pilot-phase clinical trials), the Center for Surgical Innovation (supporting ABY-029 development and IND approval) and the Center for Imaging Medicine (supporting bioengineering of fluorescence systems and pre-clinical studies). Taken together this proposal will leverage all of these resouces into the first assessment and guidance of how fluorescent molecular information could be used in guiding sarcoma surgery.